<2022-08-18 Thu> David biweekly meeting

Project 4: Reconstitution of endocytic actin network

recent progress

  • finished manually sorting through EM data for images with potential membrane structures
    • see document on Max's laptop; too much data to include here
  • started a preliminary quantification of the data
    • -Las17: 10 possible vesiculation events out of 46 beads imaged
    • +Las17: 29 possible vesiculation events out of 25 beads imaged
      • this was split over several sections though; 77 total bead slices
  • reached out to Danielle and Emily to meet and get better advice on what is actually membrane or not
    • also asked for references for methods
  • checked if we could correlate membrane budding between fluorescence and EM budding, but that doesn't seem feasible
    • vesicle I identified by EM

      2022-08-18_07-44-28_Screenshot from 2022-08-03 19-47-15.png
    • the same vesicle in context of other beads

      2022-08-18_07-45-20_Screenshot from 2022-08-03 19-51-12.png
    • the same cluster of beads by fluorescence; that vesicle is not visible

      2022-08-18_07-45-05_Screenshot from 2022-08-03 19-53-57.png

next steps

  • compare data interpretations with Emily when she finishes hers
  • quantify membrane bending/vesiculation with help from Danielle
  • write up methods for CLEM
  • refresh the manuscript

Project 7: Mechanical regulation of CME by protein LLPS

recent progress

  • bugs in my coated membrane model are mostly sorted out now; simulations are well-behaved
    • purple: coat
    • black: bare membrane
    • red: coat-membrane interface

next steps

  • try to replicate conditions of Julian's model to validate that it behaves the same
  • build final version of model with protein condensate

Nikhil's protein purification project

project overview

  1. express and purify His-tagged GFP in E. coli
  2. insert GFP into His6-eps15 plasmid from Stachowiak lab
  3. express and purify His6-eps15-GFP from E. coli
  4. purify His6-eps15
  5. purify Fcho1
  6. reproduce phase separation experiments

updates

  • while purification was successful, we weren't really observing any induction of our GFP and RFP training constructs
  • after lots of troubleshooting the culturing conditions, we realized the constructs we were working with were actually constitutive
  • decided it's not worthwhile to continue troubleshooting with the constitutive constructs, so now working on cloning GFP and GFP-EPS15 into the same vector that was used to express EPS15 in the Stachowiak lab paper
    • we engineered a cleavage site between GFP and EPS15 as a potential alternative purification strategy

Idea for theory rotation project

  • constant area vs. constant curvature models (Kaksonen and Roux 2018)

    2022-08-18_08-33-05_Screenshot from 2022-08-18 08-32-17.png
  • all my CME modeling so far is based on the assumption that the coat is fully polymerized before membrane bending starts (constant area model)
  • since the design and analysis of this simple model is already set up, I think a nice rotation project would be to modify the assumption to constant curvature instead of constant area and see if that changes the main result (initial energy barrier)
    • the geometry will be slightly different in that case, so it will be important to check
    • intuitively though, this shouldn't qualitatively change the main result, but it will probably quantitatively change it

Date: \today

Author: Max Ferrin

Email: ferrinm@berkeley.edu

Created: 2022-08-18 Thu 08:45

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